Multiciliated cells are found in abundance in the respiratory epithelium. The hundreds of motile cilia on each multiciliated cell aids in both signaling processes and mucociliary clearance. Defects in the differentiation of these cells or in cilia formation result in congenital disorders, such as primary ciliary dyskinesia (PCD), and chronic respiratory conditions, such as cystic fibrosis (CF), asthma, and chronic obstructive pulmonary disease. Despite the significant portion of the population affected by these chronic pulmonary conditions as well as the devastating nature of PCD, CF, and similar congenital disorders, little is known about the molecular mechanisms dictating multiciliated cell differentiation. In 2007, an absolute requirement in primary ciliogenesis was discovered for the ciliary protein centrosomal protein 164 (CEP164). Since this time, great interest has followed as CEP164 was revealed to be part of the distal appendage, a structure that extends from the distal portion of the centriole/basal body and is critical for vesicle recruitment and basal body docking. While prior work has made progress in uncovering the role of CEP164 in the formation of primary cilia in cultured cells and morpholino studies in zebrafish, no study has yet to examine its functionality in airway multiciliated cells in the physiological setting of a mammalian model system. The overall goal of this proposal is to define the function of CEP164 during multiciliated cell differentiation in normal and diseased airway epithelium. To accomplish this goal, this proposal employs a novel multiciliated cell-specific CEP164 knockout mouse model and a primary culture system of mouse tracheal epithelial cells (MTECs). By harnessing both of these tools, the roles of CEP164, its interacting partners, and the distal appendage will be elucidated through genetic means, biochemical assays, and direct visualization (SEM, TEM, confocal, and super-resolution microscopy). I expect that these methodologies will provide novel insights into the processes of motile ciliogenesis and multiciliated cell development. I also anticipate that, by knocking out CEP164 in multiciliated cells, a powerful mouse model of PCD will be obtained. This work will identify novel molecular networks regulating ciliogenesis and potential therapeutic targets for patients afflicted with diseases of dysfunctional motile cilia.